Materials and Methods s4

Noel D. Mueller

BioC 5025

Lab 4a – 4e

3/2/99

Purification and Characterization of Thermus aquaticus DNA Polymerase

By

Noel D. Mueller

Abstract

Escherichia coli bacterial cells containing the pT7-7 vector containing an exogenous gene from the Thermus aquaticus bacterium which encodes a heat resistant DNA polymerase (Taq enzyme) were identified in earlier work (Mueller, 1999). The Taq protein was purified using; heat extraction, ion-exchange chromatography and SDS-PAGE gel electrophoresis. Taq protein activity was maintained throughout the experimental protocol. Specific activity of the protein was found for Fraction 1(whole cell extract), Fraction II (heat extract), and Fraction III (ion-exchange chromatography extract) and commercially purified Taq protein (Amplitaqä). The protein concentrations of the three fractions was found using a BCA assay. The molecular weight of the Taq protein was found by means of SDS-PAGE gel utilizing molecular weight marker proteins. The molecular weight of Taq was found to be 87687 D. This corresponds to the protein being ~797amino acids in length. The gene which encodes a protein of this length is 2391 base pairs long. The heat extraction produced the greatest increase of the enzyme activity, from 29090units/mg for Fraction I to 84210units/mg for Fraction II. SDS-PAGE analysis of Fraction III shows it contains the most pure Taq, but a great deal of enzyme activity was lost in this purification step (specific activity of Fraction III = 19048units/mg).

Introduction

In experiment 3 (Purification and Characterization of Plasmid DNA), colonies of Escherichia coli which contained an insert of the Taq DNA polymerase gene were identified. The Taq enzyme is of great importance to the field of biochemistry because it does not denature at high temperatures (>100°). This is desirable for use in polymerase chain reaction (PCR) because the stabile Taq DNA polymerase need not be replaced after each round of heating to melt double stranded DNA to initiate DNA replication.

A pellet of E. coli cells had been induced to turn on expression of the Taq gene using IPTG. The E. coli used in this experiment were genetically engineered to express large amounts of Taq enzyme. Normally, “the proteins we are usually interested in purifying make up only !% to 0.00001% of the total protein in the starting sample”(Fuchs, 1999).

Precipitation, electrophoresis and chromatography were used to purify the Taq enzyme. Purification allows us to further analyze the properties of this protein. The molecular weight of the protein may be determined and it is possible to determine the amino acid sequence of a protein. “One of the major goals of biochemistry is to determine how amino acid sequences specify the conformations of proteins.” (Stryer, 1995). It is also desirable to purify Taq for use in PCR.

The first step in the purification of Taq enzyme was a heat extraction. Bacterial cells were lysed to obtain a crude cellular protein extract. This extract was heated which causes many proteins to denature and fall out of solution. The Taq enzyme does not denature because it is derived from the genetic material of a thermophilic bacterium.

It is important to check enzymatic activity at each step of a purification. A PCR primer-dimer assay was used to ensure that Taq activity was not lost at any step of the purification. PCR provides a means to make DNA replicate in a test tube. It works by extending a primer with DNA polymerase. A 5’ primer and a 3’ primer will form a dimer. Multiple rounds of heating, which will cause the dimer to melt (into two single strands), and a round when cooler temperatures allow new primers to anneal and are also extended. In this fashion, the DNA primer-dimers may be amplified in an exponential manner. The DNA is doubled with each round of melting and annealing. A single strand of DNA may be amplified to over 1,000,000 copies after 20 rounds in a PCR cycler. Samples containing active Taq protein will amplify the primer-dimer DNA and this result can be checked using gel electrophoresis. Gel electrophoresis works by a power source being used to create a positive and negative terminal in a gel box. DNA, which is negatively charged, will be pushed through a porous agarose gel towards the positive terminal. Using EtBr allows the DNA bands in the gel to be visualized under ultraviolet light. A unit of enzyme activity for Taq polymerase is defined as the minimal amount of Taq protein required to give a positive result (amplified DNA band visible in the agarose gel) in the primer-dimer PCR assay.

The Taq protein was further purified using ion exchange chromatography. For this experiment, a column chromatography was set up using diethyl aminoethyl (DEAE) and cellulose (an inert polymer). DEAE is positively charged at below pH 9. Different proteins have different net charges because of ionizable groups present in the protein. This fact means that different proteins will have differing affinities for the charged functional groups (DEAE). Proteins that bind to the DEAE will displace the Cl- counterion. By passing increasingly concentrated Cl- solution through the column, proteins may be eluted based upon their differential column affinities. A protein with a lesser affinity for the column is eluted at a lower salt concentration than a protein with a high affinity for the column.

The concentration of protein in solution can be found using a BCA assay. Proteins will react with Cu++ in a alkaline solution to yield Cu+. Bicinchoninic acid will turn purple in the presence of Cu+. Two molecules of BCA react with one Cu+ to yield a BCA-Cu+ complex. The purple reaction product has a strong absorbance at 562nm. A standard curve may be prepared by using known amounts of protein. Linear regression analysis of the standard allows the calculation of protein in samples where the protein concentration is not known.

A SDS-PAGE gel will allow for further characterization of the Taq enzyme. Sodium dodecylsulfate (SDS) is an anionic detergent which denatures proteins by wrapping around the polypeptide backbone (Fuchs, 1999). The SDS adds a net negative charge to the protein proportional to its length. This net negative charge on the protein will cause it to move in response to an electrical field, just as negatively charged DNA moves through an agarose gel in response to an electrical field. The SDS gel was run with molecular weight marker (MW) proteins; carbonic anhydrase (29000 D), ovalbumin (45000D), bovine serum albumin (66000 D), phosphorylase B subunit (97400 D), b-galactosidase subunit (116000 D), and myosin subunit (205000 D). A linear relationship exists between the log10 of the molecular weight and the Rf (the distance a protein migrated on the gel), which allows the molecular weight of the Taq protein to be calculated.

Lastly, the activity of the ion exchange column purified protein (Fraction III, FR III) was found by a primer-dimer PCR assay. The activity will again be defined as the minimal amount of Taq protein required to produce amplified DNA to give a positive result in the PCR assay. The activity of FR III was compared to the activity of Amplitaqä.

Materials and Methods

Heat extraction: A cell pellet of Escherichia coli cells (from 30 ml of cell culture) which had been induced to produce Taq polymerase protein by adding IPTG (to 0.5mM concentration) was obtained from laboratory staff. The pellet was resuspended using ~2ml 50mM Tris-HCl pH 7.9, 1mM EDTA. The volume of this mixture (Fraction I, FR I) was recorded for later use. 100 ml of FR I was stored at 4° for later use. An additional 3 ml of Tris Wash Buffer was added. The solution was centrifuged (6000 RPM, 10 min) and the supernate was discarded. The cells were resuspended in 1 ml Tris Wash Buffer/lysozyme. After allowing the cells to lyse for 15 minutes, 1 ml Tris Wash Buffer + detergents (10mM Tris-HCl pH 7.9, 50mM KCl, 1 mM EDTA, 0.1mM protease inhibitor, 0.5% Tween 20, 0.5% Triton X-100). The solution was heated for 60 minutes at 75°. The solution was centrifuged at 8000 RPM for 10 minutes. The supernate was collected (Fraction II, FR II) with 25ml being set aside for PCR analysis. These samples were stored at -20°.

PCR assay of FR I and FR II: A primer-dimer PCR assay of FR I and FR II was performed using the following components: 5’ primer, 1.0mM; 3’ primer, 1.0mM; dNTP’s (dATP, dCTP, dTTP, dGTP) 0.2mM each; MgCl2, 10mM; Buffer (50mM KCl, 10mM Tris, pH 8.3); and water to make volumes equal. A serial dilution (use Taq dilution buffer) was performed to give 1/20,1/40,1/80, and 1/160 dilutions of FR I and FR II. The PCR reaction was carried out with 5ml each of; undiluted FR I, undiluted FR II, the 1/20, 1/40, 1/80, 1/160 dilutions of FR I and FR II, Taq polymerase positive control and water for the negative control. 20ml of the master mix was added to each PCR reaction tube to bring the total volume to 25ml. The PCR program for this assay was 20 cycles of 15 seconds at 85° / 15 seconds at 75°. The results of the PCR assay were analyzed using a 1.2% agarose gel in 0.5X TBE buffer. 6ml of loading dye + EtBr was added to each reaction tube. The gel was run at 40 mAmps for 7-10 minutes.

Ion-exchange chromatography: A column was made using 4 ml 50% Diethyl aminoethyl (DEAE) – cellulose slurry in “column pouring buffer” (10mM Tris-HCl pH7.9, 50mM KCl, 1 mM EDTA). 50ml of FR II were saved for a protein assay and PAGE gel analysis. 6 ml “Binding Buffer” (“column pouring buffer” + 0.1 mM protease inhibitor, 0.5% Tween 20, 0.5% Triton X-100) was added to the remaining volume of FR II. This solution was poured onto the top of the column bed. The column was washed with 5 column volumes of “Binding Buffer”, with the last 1ml of the wash being collected. The protein was eluted using a Cl- gradient (150mM Buffer B-150, 200mM B-200, and 250mM KCl B-200). 4 ml of each concentration of KCl was added and 1.3ml fractions of the elutions were collected.

BCA assay: 100ml of each fraction of the ion-exchange chromatography was assayed for protein concentration. The following reagents were used: Reagent A – sodium carbonate, sodium bicarbonate, disodium BCA, and sodium tartrate in 0.2 N NaOH; Reagent B – 4% CuSO4·5H2O in deionized water; standard protein solution – Bovine Serum Albumin (BSA) at 4mg/ml of Buffer B-150; Working Reagent - 50 parts A with 1 part B. BCA assay tubes were set up as follows (Table 1).

Tube # / B-150 (ml) / Sample (ml) / Working reagent
1 / 100 / 0 / 0.9 ml
2 / 80 / 20 / 0.9
3 / 60 / 40 / 0.9
4 / 40 / 60 / 0.9
5 / 20 / 80 / 0.9
6 / 95 / 5 (FR I) / 0.9
7 / 95 / 5 (FR I !/10 dil.) / 0.9
8 / 95 / 5 (FR II) / 0.9

Table 1. BCA assay

Tubes 1-5 were used to find a standard curve for the BCA assay. Tubes 9-19 contained 100ml of the column flow-through, wash, and three collected samples from B-150, B-200, and B-250. The tubes were incubated at 37° for 30 minutes and the absorbance was read at l562 nm.

SDS-PAGE: A PCR primer-dimer assay was performed on tubes 9 (flow-through), 11 (B-150#1) – 19 (B250#3). Column fractions to be analyzed by SDS-PAGE were selected based upon results of the PCR primer-dimer assay and gel electrophoresis analysis. 30ml samples containing 10mg of protein were prepared of FR I, FR II, flow-through, and column fractions from tubes 13-16 (FR I-26.5ml, FR II-20.5ml, flow through-30ml, B150#3-30ml, B200#1-30ml, B200#2-30ml, B200#3-22.5ml). To each sample, 10ml of 4x sample buffer was added (1x sample buffer = 10% glycerol, 0.5% mercaptoethanol, 1% SDS, 50mg/ml of bromothymol blue. Samples were heated for 3 minutes in a boiling water bath. A 10% polyacrylamide gel was obtained from lab staff and a stacking gel was prepared using; ammonium persulfate, TEMED, and the stacking gel solution. 20 ml of the samples and 10ml of the molecular weight (MW) markers were added to the wells. Samples were electrophoresed at 35mA for about 45 minutes. Gels were stained with Coomassie Blue R-250 stain.

Final PCR analysis: A primer-dimer assay was performed to find the activity of Taq polymerase in FR III (tube 13, B-150#3). FR III was diluted with Taq dilution buffer to give samples of the following dilutions; 1/5, 1/10, 1,20, 1/40, 1/80, 1/160. Amplitaqä was diluted to give samples of dilutions; 1, ½, ¼, 1/8, 1/16. Results of the PCR run were analyzed by using gel electrophoresis to find the activity of FR III and Amplitaqä.

Results

The volume of Fraction I after cell lysis was found to be 3.3 ml. The volume of Fraction II after heat extraction was found to be 2.0. A primer-dimer PCR assay was performed on FR I and FR II to find the specific activity of the Taq polymerase in those two samples (Figure 1).

Figure 1. Lanes 1-5 contain Fraction I; 1-undiluted, 2-1/20, 3-1/40, 4-1/80, 5-1/160 dilutions. Lanes 6-10 contain FR II; 6-undiluted, 7-1/20, 8-1/40, 9-1/80, 10-1/160 dilutions. Lane 11 contains the positive control and 12 contains the negative control.

In both FR I and FR II, 5ml of a 1/160 dilution of the sample produced amplified DNA. The activity of these fractions was calculated. ( 5ml)(1/160 dilution) = 1 unit of activity in 0.03125 ml. ( 1ml ) / ( 0.03125 ml ) = 32 units / 1 ml. (32 units/ml)(1000ml/ml) = 32000 units of Taq activity/ml for both FR I and FR II.


Spectrophotometric analysis of the BCA assay gave the following results. A standard curve for the absorption of the BCA – Cu+ complex at 562 nm is shown in Figure 2.